1. Field of the Invention
The invention relates to methods for regulating cardiac muscle contractility. More specifically, the invention relates to methods to increase in vivo levels of cardiac sarcoplasmic reticulum (SR) calcium2++ ATPase (SERCA2) by in vivo delivery of a gene which operatively encodes SERCA2 protein.
2. History of the Prior Art
Congestive heart failure is one of the leading causes of death among adults in the United States. As compared to cardiac ischemia (an acute event resulting from obstruction or loss of blood supply to the heart), congestive heart failure is a relatively insidious event associated with the gradual loss of cardiac muscle contractility and adaptability of the heart to stress. Ultimately, absent effective treatment, the CHF heart loses its ability to pump blood at a rate sufficient to meet the metabolic requirements of the body.
Although the abnormalities in cardiac function which accompany congestive heart failure (CHF) vary, decreased release from the SR of the calcium2++ ions required for activation of contractile proteins is a common characteristic of the CHF syndrome. The significance of this loss can be best understood in the context of the role that calcium transport plays in the normal functioning of the heart.
Briefly, the SR is a membranous structure which surrounds each myofibril of cardiac muscle. SERCA2 is contained within the SR membranes and serves to actively transport 70 to 80% of free calcium ions into the SR intracellular space during diastolic relaxation of cardiac muscle. Much of the remaining calcium ions available for transport are removed from the cytoplasm by a SR sodium/calcium transport exchange system as well as, to a far lesser extent, transport driven by ATP hydrolysis CATALYZED by sarcolemma calcium ion ATPase and through mitochondrial calcium uptake (Bassani, et al., J. Physiol. 453:591-608, 1992 and Carafoli, E., Ann. Rev. Biochem., 56:395-433, 1987).
Given that both the ATP hydrolytic activity of SERCA2 and absolute levels of SERCA2 mRNA are decreased in the CHF heart (Hasenfuss, et al., Circ. Res., 75:434-442, 1994 and Studer, et al., Circ. Res. 75:443-453, 1994), it has been widely postulated that the impairment of the CHF heart""s ability to receive blood at low pressures is directly linked to delays in SERCA2 mediated transport of contraction-activating calcium ions into the SR, which in turn results in a slowing of diastolic relaxation of the heart (see, e.g., Grossman, W., N. Engl. J. Med, 325:1557-1564, 1991; Lorell, B H, Ann. Rev. Med, 42:411-436, 1991; and, Arai, et al., Circ. Res., 74:555-564, 1994). These observations, particularly with respect to reductions in levels of mRNA""s coding for SERCA2 have been confirmed in humans as well as other mammalian species (see, re human SERCA2 mRNA levels, Arai, et al., supra and Mercadier, et al., J. Clin. Invest., 85:305-309, 1990; also, re lowering of SERCA2 mRNA levels in hypertrophied heart tissue of other mammalian species, see, e.g., Wang, et al., Am. J. Physiol., 267:H918-H924, 1994 [ferrets]; Afzal and Dhella, Am. J. Physiol., 262:H868-H874, 1992 [rodents]; and, Feldman, et al., Circ. Res., 73:184-192, 1993 [rodents]).
Despite the interest in recent years regarding the role of calcium transport in CHF, the molecular basis for the impairment of SERCA2 calcium transport activity is poorly understood and has not yet been exploited in a regime for the treatment of CHF. Instead, current therapeutic modalities for CHF syndrome are largely non-specific in the sense that are not directly targeted toward the biochemical and molecular events which are believed to accompany, if not cause, the abnormalities of function which lead to failure of the CHF heart. For example, pharmaceutical treatment of CHF by administering adrenaline-like drugs stimulates cardiac muscle contraction but does not correct the underlying condition which caused the diminishment in the contractility of the muscle.
Thus, replacement and/or increase of in vivo levels of cardiac proteins is an intriguing alternative for the treatment and control of the progression of CHF in humans. However, achieving this goal by introducing cardiac proteins or peptides into heart tissue is unlikely to be successful. Of primary concern is the risk of potential toxicities, particularly at dosages sufficient to produce a biological response to the protein. From a practical perspective, there is also the problem of the cost associated with isolating and purifying or synthesizing the proteins. Moreover, the clinical impact of the proteins would also be limited by their relatively short half-life in vivo due to degradation by any proteases present in the target tissue.
For these reasons, introduction of a protein into a patient by delivery of a gene which will express the protein is an intriguing alternative to administering the protein itself. To that end, a variety of strategies have been developed for the introduction of exogenous genes into target cells. Most gene therapy protocols proposed to date for use in humans have focused on ex vivo gene transfer; e.g., by retroviral transfection of cells for implantation into target tissue (see, e.g., Anderson, W F, Science, 256:808-813, 1992 and Miller, A D, Nature, 357:455-460, 1992 [treatment of adenosine deaminase deficiency]). However, the usefulness of such protocols has proved to be limited by their relative inefficiency of protein expression as well as the limited accessibility of target organs and tissues.
In vivo gene delivery methods are therefore a topic of great interest in the art. To that end, several systems have been developed to achieve this goal, including introduction of xe2x80x9cnakedxe2x80x9d polynucleotides (plasmids), plasmids linked to viruses, plasmids cointernalized with viruses, as well as encapsulation and delivery of gene constructs within liposomes.
For example, work at the NIH was reported in 1984 which showed that intrahepatic injection of naked, cloned plasmid DNA for squirrel hepatitis into squirrels produced both viral infection and the formation of antiviral antibodies in the squirrels (Seeger, et al., Proc. Nat""l. Acad. Sci USA, 81:5849-5852, 1984). Several years later, Felgner, et al., reported that they obtained expression of protein from xe2x80x9cnakedxe2x80x9d polynucleotides (i.e., DNA or RNA not associated with liposomes or a viral expression vector) injected into skeletal muscle tissue (Felgner, et al., Science, 247:1465, 1990; see also, PCT application WO 90/11092). Felgner, et al. surmised that muscle cells efficiently take up and express polynucleotides because of the unique structure of muscle tissue, which is comprised of multinucleated cells, sarcoplasmic reticulum and a transverse tubular system which extends deep into the muscle cell.
Similar systems for delivery genes directly into target tissue have been reported by Stribling, et al., Proc. Natl. Acad. Sci. USA, 89:11277-11281, 1992 (protein expression detected after aerosol delivery of a liposome encapsulated gene); and, Tang, et al., Nature, 356:152-154, 1992 (injection with a vaccine xe2x80x9cgunxe2x80x9d of an hGH plasmid coupled to colloidal gold beads into the skin of mice resulted in hGH protein expression without eliciting an immune response to the injected gene). Although generally effective for producing high levels of protein expression within muscle cells, direct injection of DNA or RNA into muscle tissue for long-term therapy requires use of repeated injections to offset loss of expression from gene degradation. This approach may not only be time-consuming and expensive, but may also be impractical for long-term therapy due to inflammation caused at and near the site of injection. Thus, although useful in short-term or emergency therapies, less invasive means for introduction of expressible genes into target tissue will generally be preferred over direct injection into the target tissue.
Further, most methods for in vivo gene delivery free of a recombinant expression vector suffer from inefficient target cell transfection and relatively low protein expression. Thus, recombinant expression vectors (especially non-replicable vectors) presently remain the preferred vehicle for in vivo gene delivery.
Cardiac myocytes have been shown to be suitable targets for in vivo gene delivery. For example, one recent proposal for treatment of CHF would replace and/or enhance the numbers of active xcex22-adrenergic receptors in myocytes of the CHF heart. Studies in mice have indicated that use of direct transplantation techniques to introduce genes which encode such receptors leads to increased in vivo contractility of the heart muscle even in the absence of an exogenous adrenaline source (Lefkowitz, et al., Science, 264:582-586, 1994). Adenovirus vectors in particular have been shown to be successful vehicles for delivery of genes to cardiac myocytes (see, e.g., Guzman, et al., Circ. Res., 73:1202-1207, 1993 and Muhlhauser, et al., Circulation, 88(Part 2):1-475, 1993).
The details of the preferred embodiment of the present invention are set forth in the accompanying drawings and the description below. Once the details of the invention are known, numerous additional innovations and changes will become obvious to one skilled in the art.
The invention is a method for augmenting calcium transport into the SR of heart muscle by elevating in vivo expression of catalytically active SERCA2 by introducing a polynucleotide which operatively encodes SERCA2 into such tissue. In an embodiment preferred for its efficiency of target cell transfection, the SERCA2 encoding polynucleo-tide is delivered into heart tissue via a non-replicable viral recombinant expression vector, most preferably an adenovirus construct. Alternatively, the SERCA2 encoding gene may be provided by encapsulation in liposomes or by administration of xe2x80x9cnakedxe2x80x9d nucleotides. Delivery of the SERCA2 gene may be made surgically (i.e., by direct introduction of the vector or transfected cells into target tissue) or by intracoronary infusion.
The enhancement in transport of calcium ions to the SR provided by increased SERCA2 activity in the SR will moderate activation of myofibril contractions in the heart. Thus, a particular advantage of the method of the invention is the assistance it will provide the heart in adjusting to the abnormalities associated with CHF, especially by shortening the early phase of diastolic relaxation of heart muscle.
One use for the method of the invention is for short-term amelioration of CHF in patients who are awaiting interventional therapy (e.g., transplantation) and/or who are already undergoing treatment for CHF. Use of the method of the invention is also expected to benefit patients who are at risk for, and have begun to suffer from, the abnormalities in heart function associated with CHF.
In another aspect of the invention, transgenic animals are provided for use in developing vectors for use in, and testing the application of, the method of the invention. Methods for monitoring the efficacy of the method of the invention as used for treatment of CHF are also described.